The long-term goal of this research is to identify mechanisms required for new blood vessel formation (e.g. angiogenesis). The basic shape changes that endothelial cells (ECs) undergo during angiogenesis, including vacuole and lumen formation, and branching morphogenesis are areas that need more investigation. Using in vitro systems developed in our laboratory, human BC resuspended in both collagen and fibrin matrices undergo morphogenesis. This process involves the formation of vacuoles that then branch to form interconnected networks over time. These dramatic shape changes require the actin cytoskeleton. The Rho family of GTPases have been reported to regulate cell shape changes controlled by the actin cytoskeleton. The first aim of the proposal is to study the involvement of RhoA, Rac 1 and Cdc42 in EC lumen formation in three-dimensions. To determine the individual roles of each GTPase in vacuole and lumen formation, recombinant adenoviruses were constructed to manipulate gene expression in ECs. To address this question, adenoviruses expressing dominant negative and constitutively active forms of RhoA, Rac 1 and Cdc42 were constructed. The ability of these viruses to block vacuole and lumen formation, and branching morphogenesis will be determined. In addition, adenoviruses will deliver GFP-Rho GTPase chimeras to determine where these molecules target in ECs at the distinct steps of morphogenesis. Based on these findings, we will screen for both novel and known binding partners of Rho GTPases in ECs using a yeast two-hybrid cDNA library from ECs and also immunoaffinity chromatography. Further, overexpression of dominant negative downstream effectors for the Rho GTPases will be used to further dissect downstream signaling events in ECs that control the different steps in morphogenesis. Discovering the basic mechanism of EC vacuole and lumen formation, and branching morphogenesis within three-dimensional collagen and fibrin matrices may help uncover fundamental mechanisms required for blood vessel formation by endothelial cells.